Novel activin receptor and uses thereof

ABSTRACT

The present invention provides novel activin IIB5 receptor polypeptides capable of binding and inhibiting the activities of activin A, myostatin, or GDF-11. The present invention also provides polynucleotides, vectors and host cells capable of producing the receptor polypeptides. Compositions and methods for treating muscle-wasting, metabolic and other disorders are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 60/732,270, filed Nov. 1, 2005, U.S. Ser. No. 11/590,962, filed Oct.31, 2006 (now U.S. Pat. No. 8,067,562), and U.S. Ser. No. 13/190,255,filed Jul. 25, 2011, the entire disclosure of each of which is reliedupon and incorporated herein by reference, in its entirety, for allpurposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Nov. 19, 2013, is named25010US_sequencelisting.txt, and is 21,902 bytes in size.

TECHNICAL FIELD OF THE INVENTION

The technical field of this invention relates to transforming growthfactor-β (TGF-β) family members and TGF-β receptors, as well as methodsof modulating the activities of TGF-β family members for the treatmentof various disorders.

BACKGROUND OF THE INVENTION

The transforming growth factor β (TGF-β) family of proteins includes thetransforming growth factors-β (TGF-β), activins, bone morphogenicproteins (BMP), nerve growth factors (NGFs), brain-derived neurotrophicfactor (BDNF), and growth/differentiation factors (GDFs). These familymembers are involved in the regulation of a wide range of biologicalprocesses including cell proliferation, differentiation, and otherfunctions. Activins were originally discovered as gonadal peptidesinvolved in the regulation of follicle stimulating hormone synthesis,and are now believed to be involved in the regulation of a number ofbiological activities including control of section and expression ofanterior pituitary hormones such as FSH, GH, and ACTH, neuron survival,hypothalamic oxytocin secretion, erythropoiesis, placental and gonadalsteroidogenesis, early embryonic development, and proliferation of sometypes of tumors. Activins A, B and AB are the homodimers and heterdimerrespectively of two polypeptide chains, βA and βB (Vale et al. Nature321, 776-779 (1986), Ling et al., Nature 321, 779-782 (1986)). These twoβ chains can also dimerize with a related a chain giving rise toinhibins A and B respectively (Mason et al, Nature 318, 659-663 (1986)).It is well established that inhibins are necessary for maintainingnormal function in many tissues, particularly those associated withreproductive functions. In these tissues inhibins oppose many, but notall, of the activin activities.

Growth/differentiation factor 8 (GDF-8), also referred to as myostatin,is a TGF-β family member expressed for the most part in the cells ofdeveloping and adult skeletal muscle tissue. Myostatin appears to playan essential role in negatively controlling skeletal muscle growth(McPherron et al. Nature (London) 387, 83-90 (1997)). Antagonizingmyostatin has been shown to increase lean muscle mass in animals(McFerron et al, supra, Zimmers et al, Science 296:1486 (2002)).

Another member of the TGF-β family of proteins is a relatedgrowth/differentiation factor, GDF-11. GDF-11 has approximately 90%identity of the amino acid sequence of myostatin. GDF-11 has a role inthe axial patterning in developing animals (Oh et al, Genes Dev11:1812-26 (1997)), and also appears to play a role in skeletal muscledevelopment and growth. However, the postnatal role of GDF-11 is notcurrently understood.

A family of transmembrane serine/threonine kinases are known to act asreceptors for activins and other TGF-β family members. These receptorsfall into two distinct subfamilies known as type I and type II receptorsthat act cooperatively to bind ligand and transduce signal (Attisano etal., Mol Cell Biol 16 (3), 1066-1073 (1996)). Most TGF-β ligands arebelieved bind first to a type II receptor and this ligand/type IIreceptor complex then recruits a type I receptor (Mathews, L S, EndocrRev 15:310-325 (1994); Massague, Nature Rev: Mol Cell Biol. 1, 169-178(2000)). The type II receptor kinase then phosphorylates and activatesthe type I receptor kinase, which in turn phosphorylates the Smadproteins. Activins initially bind their type II receptors ActRIIA foractivin A, or ActRIIB for activin B. This is followed by therecruitment, phosphorylation and subsequent activation of the type Ireceptor, activin-like kinase 4 (ALK4). On activation, ALK4 binds andthen phosphorylates a subset of cytoplasmic Smad proteins (Smad2 andSmad3) that produce signal transduction for activins (Derynck, R et al.Cell 95, 737-740 (1998)).

Cross-linking studies have determined that myostatin is capable ofbinding the activin type II receptors ActRIIA and ActRIIB in vitro (Leeet al. PNAS USA 98:9306-11 (2001)). There is also evidence that GDF-11binds to both ActRIIA and ActRIIB (Oh et al., Genes Dev 16:2749-54(2002)).

TGF-β proteins are known to be associated with a variety of diseasestates and antagonizing these proteins may be useful as therapeutictreatments for the disease states. In particular antagonizing severalTGF-β proteins simultaneously may be particularly effective for treatingcertain diseases. The present invention provides a novel composition ofmatter and methods of using the composition of matter as a treatment formuscle-related and other disorders.

SUMMARY OF THE INVENTION

The present invention provides a protein comprising human activinreceptor IIB5 (designated ActRIIB5) polypeptides. In one embodiment, theprotein comprises polypeptides having an amino acid sequence set forthin SEQ ID NO: 2. In another embodiment the protein comprises apolypeptide having an amino acid sequence with at least about 80% orgreater identity to SEQ ID NO: 2, wherein the polypeptide is capable ofbinding myostatin, activin A, or GDF-11. In another embodiment, theprotein comprises a polypeptide having an amino acid sequence with atleast about 80% or greater identity to SEQ ID NO: 2, wherein the Cterminal of the polypeptide consists of the amino acid sequence setforth in SEQ ID NO: 3, and wherein the polypeptide is capable of bindingmyostatin, activin A, or GDF-11. In another embodiment, the proteincomprises a polypeptide having an amino acid sequence with at leastabout 80% or greater identity to SEQ ID NO: 2, wherein the C terminal ofthe polypeptide has an amino acid sequence with at about least 80% orgreater identity to SEQ ID NO: 3, and wherein the polypeptide is capableof binding myostatin, activin A, or GDF-11. In one embodiment, thepolypeptide lacks an ActRIIB5 signal sequence. In another embodiment,the protein comprises a polypeptide encoded by the polynucleotide havingthe sequence set forth in SEQ ID NO: 1.

In another embodiment, the protein of the present invention comprisesActRIIB5 polypeptides fused to one or more heterologous polypeptides. Inone embodiment, the fused ActRIIB5 polypeptides lack a signal sequence.In one embodiment the ActRIIB5 polypeptides are fused to theheterologous polypeptides via one or more linker sequences. In anotherembodiment the heterologous polypeptides comprise an Fc domain. Inanother embodiment, the Fc domain is connected to the ActRIIB5polypeptides by at least one linker sequence. In another embodiment,ActRIIB5 polypeptides are attached to a non-protein carrier moleculesuch as a PEG molecule.

In another aspect the present invention provides an isolated nucleicacid molecule comprising a polynucleotide encoding an ActRIIB5polypeptide. In one embodiment, the nucleic acid molecule comprises (a)a polynucleotide having the nucleic acid sequence set forth in SEQ IDNO: 1 or its complement. In another embodiment, the nucleic acidmolecule comprises (b) a polynucleotide encoding a polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO: 2 or itscomplement. In another embodiment, the nucleic acid molecule comprises(c) a polynucleotide which hybridizes to (a) or (b) under conditions ofat least moderate stringency in about 50% formamide, 6×SSC at about 42°C. and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS, andwherein the encoded polypeptide comprises a C terminal having an aminoacid sequence set forth in SEQ ID NO: 3, and wherein the polypeptide iscapable of binding myostatin, activin A, or GDF-11. In anotherembodiment, the nucleic acid molecule comprises the polynucleotide of(c) wherein the C terminal of the encoded polypeptide has an aminosequence at least about 80% or greater identity to SEQ ID NO: 3, andwherein the polypeptide is capable of binding myostatin, activin A, orGDF-11. In another embodiment, the nucleic acid molecule comprises apolynucleotide having at least about 80% or greater identity to SEQ IDNO: 1.

In another embodiment, the nucleic acid molecule of the presentinvention further comprises polynucleotides encoding at least oneheterologous protein in frame with the polynucleotides encoding anActRIIB5 polypeptide. In one embodiment, nucleic acid molecule comprisespolynucleotides encoding linker peptide sequences attaching the ActRIIB5polypeptide to at least one heterologous protein. In another embodimentthe heterologous protein is an Fc polypeptide. The present inventionfurther provides a vector comprising the nucleic acid molecules setforth above, as well as a host cell genetically engineered to expressthe nucleic acid molecules described above, and methods for producingthe ActIIRB5 protein.

The present invention further provides a composition containing theprotein of the present invention. In one embodiment, the composition isa pharmaceutical composition containing the protein in admixture with apharmaceutically acceptable carrier.

In another aspect, the present invention provides a method of inhibitingthe TGF-β proteins myostatin, activin or GDF-11 activity in vitro and invivo by contacting the proteins with an ActRIIB5 polypeptide. In anotheraspect the present invention provides a method of increasing lean musclemass and strength, and a method of increasing the ratio of lean muscleto fat in a subject in need thereof by administering an effective amountof the composition containing ActRIIB5 proteins to the subject. In oneembodiment of this method, the subject is a food animal.

In another aspect, the present invention provides a method of treatingor preventing a muscle wasting disease in a subject suffering from sucha disorder by administering a therapeutic composition containing anActRIIB5 protein to the subject. The muscle wasting disease includes orresults from, but is not limited to, the following conditions: musculardystrophy, amyotrophic lateral sclerosis, congestive obstructivepulmonary disease, chronic heart failure, cancer cachexia, chemicalcachexia, HIV/AIDS, renal failure, uremia, rheumatoid arthritis,age-related sarcopenia, organ atrophy, carpal tunnel syndrome, androgendeprivation, and muscle-wasting due to inactivity such as prolonged bedrest, spinal chord injury, stroke, bone fracture, aging. The musclewasting may also result from events such as weightlessness from spaceflight, insulin resistance, muscle wasting due to burns, androgendeprivation, and other disorders. In another aspect, the presentinvention provides a method of treating a disease correlated toexpression of activin A. In one embodiment, the disease is cancer. Inanother aspect, the present invention provides a method of treating ametabolic disorder comprising administering a therapeutic composition toa subject in need of such treatment, wherein the metabolic disorder isselected from diabetes, obesity, impaired glucose tolerance,hyperglycemia, androgen deprivation, metabolic syndrome, and bone loss.In another aspect, the present invention provides a method of genetherapy comprising administering a vector encoding the ActRIIB5 proteinsof the present invention protein to a subject in need thereof, whereinthe vector is capable of expressing the ActRBII5 polypeptide in thesubject.

The present invention further provides a method of detecting andquantitating the TGF-β proteins myostatin, GDF-11 or activin A bycontacting these proteins with an ActRIIB5 polypeptide and detecting thepolypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of Biacore® assay determination of EC₅₀ forActRIIB5/Fc compared to ActRIIB ECD/Fc.

FIG. 2 shows the increase in body weight over time in C57B1/6 miceinjected with AAV-activin A, AAV-promyostatin/Fc, AAV-ActRIIB5/Fc,AAV-ActRIIB-ECD/Fc and AAV-empty vector control.

FIG. 3 shows the percentage of body weight change compared to thecontrol at seven weeks post viral infection in C57B1/6 mice injectedwith AAV-activin A, AAV-ActRIIB5/Fc, AAV-ActRIIB-ECD/Fc, andAAV-promyostatin/Fc vector.

FIG. 4A shows a decrease in body weight over time for Ay obese miceinjected with AAV-ActRIIB5/Fc compared with a control group of Ay obesemice injected with AAV-empty vectors over a period of about threemonths. FIG. 4B shows a decrease in weekly food intake for the samegroup of AAV-ActRIIB5 mice compared with the control group over the sameperiod of time.

FIG. 5A shows the change in lean body mass over time for Ay obese miceinjected with AAV-ActRIIB5/Fc compared with a control group of Ay obesemice injected with AAV-empty vector over a period of about three months.FIG. 5B shows a large decrease in fat mass for the AAV-ActRIIB5/Fc micecompared with a control group of AAV-empty mice over the same period oftime.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel human activin receptor designatedactivin receptor IIB5 (ActRIIB5). This receptor is characterized by itsability to bind to three TGF-β proteins, myostatin (GDF-8), activin A,and GDF-11, and to inhibit the activities of these proteins.

As used herein the term “TGF-β family members” or “TGF-β proteins”refers to the structurally related growth factors of the transforminggrowth factor family including activins, and growth and differentialfactor (GDF) proteins (Kinglsey et al. Genes Dev. 8: 133-146 (1994),McPherron et al. Growth factors and cytokines in health and disease,Vol. 1B, D. LeRoith and C. Bondy. ed., JAI Press Inc., Greenwich, Conn.,USA: pp 357-393). GDF-8, also referred to as myostatin, is now know tobe a negative regulator of skeletal muscle tissue (McPherron et al. PNASUSA 94:12457-12461 (1997)). Myostatin is synthesized as an inactivepreproprotein complex approximately 375 amino acids in length, havingGenBank Accession No: AAB86694 for human. The precursor protein isactivated by proteolytic cleavage at a tetrabasic processing site toproduce an N-terminal inactive prodomain and an approximately 109 aminoacid C-terminal protein which dimerizes to form a homodimer of about 25kDa. This homodimer is the mature, biologically active protein (Zimmerset al., Science 296, 1486 (2002)). As used herein, the term “prodomain”or “propeptide” refers to the inactive N-terminal protein which iscleaved off to release the active C-terminal protein. As used herein theterm “myostatin” or “mature myostatin” refers to the mature,biologically active C-terminal polypeptide, in monomer, dimer or otherform, as well as biologically active fragments or related polypeptidesincluding allelic variants, splice variants, and fusion peptides andpolypeptides. The mature myostatin has been reported to have 100%sequence identity among many species including human, mouse, chicken,porcine, turkey, and rat (Lee et al., PNAS 98, 9306 (2001)). As usedherein GDF-11 refers to the BMP protein having Swisspro accession numberO95390, as well as variants and species homologs of that protein. GDF-11has approximately 90% identity to myostatin at the amino acid level.GDF-11 is involved in the regulation of anterior/posterior patterning ofthe axial skeleton (McPherron et al, Natr Genet 22 (93): 260-264 (1999);Gamer et al, Dev. Biol. 208 (1), 222-232 (1999)) but postnatal functionsare unknown. Activin A is the homodimer of the polypeptide chains BA. Asused herein the term “activin A” refers to the activin protein havingGenBank Accession No: NM_(—)002192, as well as variants and specieshomologs of that protein.

Activin Receptors

As used herein, the term “activin type II B receptor” (ActRIIB) refersto the human precursor activin receptor having accession numberNP_(—)001097 for protein or any variants or homologs of this receptor.The human ActRIIB precursor polynucleotide and amino acid sequences areset forth in SEQ ID NO: 4 and 5 respectively. A variation of ActRIIB isset forth in SEQ ID NO: 6, wherein arginine at position 64 has beenreplaced with alanine. SEQ ID NO: 5 is referred to as the R form and SEQID NO: 6 is referred to as the A form. The extracellular domain ofActRIIB (ActRIIB-ECD) is represented by amino acids 1 through 124 of SEQID NO: 5 and 6. Additional murine isoforms for this receptor have beenidentified as muActRIIB1, muActRIIB2, muActRIIB3 and muActRIIB4.

The present invention provides a novel human activin receptor designatedactivin receptor IIB5 (ActRIIB5). This receptor is characterized by theC terminal sequence set forth in SEQ ID NO: 3. The cDNA of this receptorwas isolated as described in Example 1, and was found to be missing the152 nucleotide bases corresponding to exon 4. This receptor is furthercharacterized as missing the transmembrane region encoded by exon 4 ofthe ActRIIB. This receptor is further characterized as being a soluble,secreted instead of a membrane bound receptor. The receptor is furthercharacterized as having the ability to bind and inhibit the activity ofany one of activin A, myostatin, or GDF-11.

The present invention provides isolated proteins which comprise ActIIB5receptor polypeptides. As used herein the term “isolated” refers to anucleic acid molecule purified to some degree from endogenous material.In one embodiment, the protein comprises ActRIIB5 polypeptides havingthe amino acid sequence set forth in SEQ ID NO: 2, and variants andderivatives of this polypeptide, which retain the activity of thepolypeptide of SEQ ID NO: 2. In one embodiment, the protein comprises apolypeptide having at least about 80% identity, at least about 85%identity, at least about 90% identity, at least about 95% identity, atleast about 98% identity, or at least about 99% identity to the aminoacid sequence set forth in SEQ ID NO: 2, wherein the polypeptide retainsthe activity of the polypeptide of SEQ ID NO: 2. In another embodiment,the protein comprises the ActRIIB5 polypeptides described above whereinthe polypeptide has a C terminal comprising the amino acid sequence setforth in SEQ ID NO: 3, and wherein the polypeptide retains the activityof the polypeptide of SEQ ID NO: 2. In another the embodiment, theprotein comprises the ActRIIB5 polypeptides described above wherein theC terminal has an amino acid sequence having at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, or at least about 99% identity to SEQ ID NO: 3, wherein thepolypeptide retains the activity of the polypeptide of SEQ ID NO: 2. Inone embodiment, the ActRIIB5 polypeptide lacks a signal sequence of SEQID NO: 2, for example, amino acids 1 to 17 of SEQ ID NO: 2.

As used herein the term “variant” refers a polypeptide having one ormore amino acid inserted, deleted or substituted into the original aminoacid sequence, but having a sequence which remains substantially similarto SEQ ID NO: 2, and which retain the activities of ActRIIB5polypeptides SEQ ID NO: 2. As used herein fragments of the polypeptideswhich retain the activity of the polypeptides are included in the term“variants”. For the purposes of the present invention, “substantiallysimilar” is at least about 80% identical to the amino acid sequence, atleast about 85% identical, at least about 90% identical, at least about95% identical, at least about 98% identical, at least about 99%identical to the amino acid sequence set forth in SEQ ID NO: 2, andretain the biological activities of the polypeptide of SEQ ID NO 2 Aminoacid substitutions which are conservative substitutions are unlikely toaffect biological activity are considered identical for purposes of thisinvention and include the following: Ala for Ser, Val for Ile, Asp forGlu, Thr for Ser, Ala for Gly, Ala for Thr, Ser for Asn, Ala for Val,Ser for Gly, Tyr for Phe, Ala for Pro, Lys for Arg, Asp for Asn, Leu forIle, Leu for Val, Ala for Glu, Asp for Gly, and the reverse. (See, forexample, Neurath et al., The Proteins, Academic Press, New York (1979)).Additional information regarding phenotypically silent amino acidexchanges can be found in Bowie et al., 1999, Science 247:1306-1310.Amino acid substitutions also include substitutions in SEQ ID NO: 2 ofnon-naturally occurring amino acids, D-amino acids, altered amino acids,or peptidomimetics. Amino acid substitutions also includesnon-conservative amino acid substitutions, such as neutral hydrophobicfor neutral polar, acidic for basic, and other class substitutions,provided that the substituted polypeptides retain the activities of thepolypeptides having the amino acid sequence in SEQ ID NO: 2. Variantsfurther include modifications to the C and N termini which arise fromprocessing due to expression in various cell types such as mammaliancells, E. coli, yeasts and other recombinant host cells. Variantsfurther include polypeptide fragments and polypeptides comprisinginactivated N-glycosylation site(s), inactivated protease processingsite(s), or conservative amino acid substitution(s), of the polypeptidesequence set forth in SEQ ID NO: 2.

Identity and similarity of related peptides and polypeptides can bereadily calculated by known methods. Such methods include, but are notlimited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork (1993); Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,and Griffin, H. G., eds. Humana Press, New Jersey (1994); SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press (1987);Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M.Stockton Press, New York (1991); and Carillo et al., SIAM J. AppliedMath., 48:1073 (1988). Methods of determining the relatedness or percentidentity of two polypeptides are designed to give the largest matchbetween the sequences tested. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package, including GAP (Devereux et al., Nucl. Acid.Res., 12:387 (1984); Genetics Computer Group, University of Wisconsin,Madison, Wis., BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol.Biol., 215:403-410 (1990)). The BLASTX program is publicly availablefrom the National Center for Biotechnology Information (NCBI) and othersources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;Altschul et al., supra (1990)). The well-known Smith Waterman algorithmmay also be used to determine identity.

As used herein the term “derivative” of the ActRIIB5 polypeptides refersto the attachment of at least one additional chemical moiety, or atleast one additional polypeptide to form covalent or aggregateconjugates such as glycosyl groups, lipids, acetyl groups, or C-terminalor N-terminal fusion proteins, conjugation to PEG molecules, and othermodifications which are described more fully below.

As used herein, the term an “ActRIIB5 polypeptide activity” or “abiological activity of ActRIIB5 polypeptide” refers to one or more invitro or in vivo activities of the ActRIIB5 polypeptides including butnot limited to those demonstrated in the Examples below. Activities ofthe ActRIIB5 polypeptides include, but are not limited to, the abilityto bind to myostatin or activin A or GDF-11, the ability to reduce orneutralize an activity of myostatin or activin A or GDF-11. For example,pMARE C2C12 cell-based assay described in Example 3 below measuresactivin A neutralizing activity, myostatin neutralizing activity, andGDF-11 neutralizing activity. In vivo activities include but are notlimited to increasing body weight, increasing lean muscle mass, anddecreasing fat mass as demonstrated in animal models below. Biologicalactivities further include reducing or preventing cachexia caused bycertain types of tumors, and preventing metastasis of certain tumorcells. Further discussion of ActRIIB5 polypeptide activities is providedbelow.

The proteins of the present invention further comprise heterologousproteins attached to the ActRIIB5 polypeptide either directly or througha linker sequence to form a fusion protein. As used herein the term“fusion protein” refers to a protein having a heterologous polypeptideattached via recombinant DNA techniques. Heterologous proteins includebut are not limited to Fc polypeptides, his tags, and leucine zipperdomains to promote oligomerization and stabilization of the ActRIIB5polypeptides as described in, for example, WO 00/29581, which is hereinincorporated by reference. As used herein the term “Fc” or “Fcpolypeptide” refers to polypeptides containing the Fc domain of anantibody. The “Fc domain” refers to the portion of the antibody that isresponsible for binding to antibody receptors on cells. An Fc domain cancontain one, two or all of the following: the constant heavy 1 domain(C_(H)1), the constant heavy 2 domain (C_(H)2), the constant heavy 3domain (C_(H)3), and the hinge region. The Fc domain of the human IgG1,for example, contains the C_(H)2 domain, and the C_(H)3 domain and hingeregion, but not the C_(H)1 domain. Truncated forms of such polypeptidescontaining the hinge region that promotes dimerization are alsoincluded. See, for example, C. A. Hasemann and J. Donald Capra,Immunoglobins: Structure and Function, in William E. Paul, ed. One Fc isa fully human Fc which may originate from any of the immunoglobulins,such as IgG1 and IgG2. However, Fc molecules that are partially human,or originate from non-human species are also included herein. Fcmolecules may be made up of monomeric polypeptides that may be linkedinto dimeric or multimeric forms by covalent (i.e., disulfide bonds) andnon-covalent association. The number of intermolecular disulfide bondsbetween monomeric subunits of native Fc molecules ranges from 1 to 4depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2,IgG3, IgA1, IgGA2). The term “Fc” as used herein is used to refer to themonomeric, dimeric, and multimeric forms. As used herein, the term “Fcvariant” refers to a modified form of a native Fc sequence. Fc variantsmay be constructed for example, by substituting or deleting residues,inserting residues or truncating portions containing the site. Theinserted or substituted residues may also be altered amino acids, suchas peptidomimetics or D-amino acids.

The proteins of the present invention can optionally further comprise a“linker” group. Linkers serve primarily as a spacer between apolypeptide and a second heterologous protein or other type of fusion orbetween two or more ActRIIB5 polypeptides. In one embodiment, the linkeris made up of amino acids linked together by peptide bonds, preferablyfrom 1 to 20 amino acids linked by peptide bonds, wherein the aminoacids are selected from the 20 naturally occurring amino acids. One ormore of these amino acids may be glycosylated, as is understood by thosein the art. In one embodiment, the 1 to 20 amino acids are selected fromglycine, alanine, proline, asparagine, glutamine, and lysine.Preferably, a linker is made up of a majority of amino acids that aresterically unhindered, such as glycine and alanine. Exemplary linkersare polyglycines (particularly (Gly)₅, (Gly)₈, poly(Gly-Ala), andpolyalanines.

The linkers of the present invention are also non-peptide linkers. Forexample, alkyl linkers such as —NH—(CH₂)s-C(O)—, wherein s=2-20 can beused. These alkyl linkers may further be substituted by anynon-sterically hindering group such as lower alkyl (e.g., C₁-C₆) loweracyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc.

The proteins of the present invention can also be attached to anon-protein molecule for the purpose of conferring desired propertiessuch as reducing degradation and/or increasing half-life, reducingtoxicity, reducing immunogenicity, and/or increasing the biologicalactivity of the ActRIIB polypeptides. Exemplary molecules include butare not limited to linear polymers such as polyethylene glycol (PEG),polylysine, a dextran; a lipid; a cholesterol group (such as a steroid);a carbohydrate, or an oligosaccharide molecule.

In another aspect, the present invention provides isolated nucleic acidmolecules comprising polynucleotides encoding the ActRIIB5 polypeptidesof the present invention. As used herein the term “isolated” refers tonucleic acid molecules purified to some degree from endogenous material.In one embodiment, the nucleotide acid molecule of the present inventioncomprises a polynucleotide encoding SEQ ID NO: 2. Due to the knowndegeneracy of the genetic code, wherein more than one codon can encodethe same amino acid, a DNA sequence can vary from that shown in SEQ IDNO: 1, and still encode a polypeptide having the amino acid sequence ofSEQ ID NO: 2. Such variant DNA sequences can result from silentmutations occurring during production, or can be the product ofdeliberate mutagenesis of SEQ ID NO: 2. In another embodiment thenucleic acid molecule comprises a polynucleotide encoding a polypeptidehaving at least about 80% identity to SEQ ID NO: 2, at least about 90%identity to SEQ ID NO: 2, at least about 95% identity to SEQ ID NO: 2,at least about 99% identity to SEQ ID NO: 2.

The percent identity may be determined by visual inspection andmathematical calculation. Alternatively, the percent identity of twonucleic acid sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described by(Devereux et al., Nucl. Acids Res., 12:387 (1984)) and available fromthe University of Wisconsin Genetics Computer Group (UWGCG). Thepreferred default parameters for the GAP program include: (1) acomparison matrix (containing a value of 1 for identities and 0 fornon-identities) for nucleotides, and the weighted comparison matrix of(Gribskov and Burgess, Nucl. Acids Res., 14:6745 (1986)), as describedby (Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358 (1979)); (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps. Other programs usedby one skilled in the art of sequence comparison may also be used.

In another embodiment the nucleic acid molecule of the present inventioncomprises a polynucleotide having the polynucleotide sequence set forthin SEQ ID NO: 1, or the complementary strand of SEQ ID NO: 1. In anotherembodiment, the present invention provides nucleic acid molecules whichhybridize under stringent or moderate conditions with thepolypeptide-encoding regions of SEQ ID NO: 1, wherein the encodedpolypeptide comprises a C terminal amino acid sequence as set forth inSEQ ID NO: 3, and wherein the encoded polypeptide maintains an activityof ActRIIB5 polypeptides.

In another embodiment, the present invention provides nucleic acidmolecules which hybridize under stringent or moderate conditions withthe polypeptide-encoding regions of SEQ ID NO: 1, wherein the encodedpolypeptide comprises a C terminal amino acid sequence having at leastabout 80% identity, at least about 85% identity, at least about 90%identity, at least about 95% identity, at least about 98% identity, atleast about 99% identity to the amino acid sequence set forth in SEQ IDNO: 3, and wherein the encoded polypeptide has at least one activity ofActRIIB5 polypeptides.

As used herein, conditions of moderate stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth by(Sambrook et al. Molecular Cloning: A Laboratory Manual, 2ed. Vol. 1,pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989)), and includeuse of a prewashing solution for the nitrocellulose filters 5×SSC, 0.5%SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50%formamide, 6×SSC at about 42° C. (or other similar hybridizationsolution, such as Stark's solution, in about 50% formamide at about 42°C.), and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS.Conditions of high stringency can also be readily determined by theskilled artisan based on, for example, the length of the DNA. Generally,such conditions defined as “highly stringent conditions” forhybridization and washing are 0.015 M sodium chloride, 0.0015 M sodiumcitrate at 65-68° C. or 0.015 M sodium chloride, 0.0015 M sodiumcitrate, and 50% formamide at 42° C. Other conditions includehybridizing and washing at approximately 68° C., 0.2×SSC, 0.1% SDS. Theskilled artisan will recognize that the temperature and wash solutionsalt concentration can be adjusted as necessary according to factorssuch as the length of the sequence. See Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory, 1989); Anderson et al., Nucleic Acid Hybridisation: APractical Approach, Ch. 4 (IRL Press Limited).

Nucleic acid molecules of the invention include DNA in bothsingle-stranded and double-stranded form, as well as the RNA complementthereof. DNA includes, for example, cDNA, genomic DNA, synthetic DNA,DNA amplified by PCR, and combinations thereof. Genomic DNA may beisolated by conventional techniques, such as by using the cDNA of SEQ IDNO:1, or a suitable fragment thereof, as a probe. Genomic DNA encodingActRIIB5 polypeptides is obtained from genomic libraries which areavailable for a number of species. Synthetic DNA is available fromchemical synthesis of overlapping oligonucleotide fragments followed byassembly of the fragments to reconstitute part or all of the codingregions and flanking sequences. RNA may be obtained from procaryoticexpression vectors which direct high-level synthesis of mRNA, such asvectors using T7 promoters and RNA polymerase. cDNA is obtained fromlibraries prepared from mRNA isolated from various tissues that expressActRIIB5. The DNA molecules of the invention include full length genesas well as polynucleotides and fragments thereof. The full length genemay also include sequences encoding the N-terminal signal sequence.

The invention also provides methods of producing and identifyingActRIIB5 polynucleotides. The well-known polymerase chain reaction (PCR)procedure may be employed to isolate and amplify a DNA sequence encodinga desired protein fragment. Oligonucleotides that define the desiredtermini of the DNA fragment are employed as 5′ and 3′ primers. Theoligonucleotides may additionally contain recognition sites forrestriction endonucleases, to facilitate insertion of the amplified DNAfragment into an expression vector. PCR techniques are described inSaiki et al., Science, 239:487 (1988); Wu et al., Recombinant DNAMethodology, eds., Academic Press, Inc., San Diego, pp. 189-196 (1989);and Innis et al., PCR Protocols: A Guide to Methods and Applications,eds., Academic Press, Inc. (1990).

In another aspect of the present invention, expression vectorscontaining the nucleic acid sequences are also provided, and host cellstransformed with such vectors and methods of producing the ActRIIB5polypeptides are also provided. The term “expression vector” refers to aplasmid, phage, virus or vector for expressing a polypeptide from apolynucleotide sequence. Vectors for the expression of ActRII5polypeptides contain at a minimum sequences required for vectorpropagation and for expression of the cloned insert. An expressionvector comprises a transcriptional unit comprising an assembly of (1) agenetic element or elements having a regulatory role in gene expression,for example, promoters or enhancers, (2) a sequence that encodesActRIIB5 polypeptides to be transcribed into mRNA and translated intoprotein, and (3) appropriate transcription initiation and terminationsequences. These sequences may further include a selection marker.Vectors suitable for expression in host cells are readily available andthe nucleic acid molecules are inserted into the vectors using standardrecombinant DNA techniques. Such vectors can include promoters whichfunction in specific tissues, and viral vectors for the expression ofActRIIB5 in targeted human or animal cells. Some exemplary expressionvectors suitable for expression of ActRIIB5 include, but are not limitedto, pDSRa, (described in WO 90/14363, herein incorporated by reference)and its derivatives, containing ActRIIB5 polynucleotides, and pDC323 orpDC324 vectors (described in Bianchi et al, Biotech and Bioengineering,Vol 84(4):439-444 (2003)) containing ActRIIB5 polynucleotides, as wellas additional suitable vectors known in the art or described below, areprovided by the present invention.

The application further provides methods of making ActRIIB5 polypeptidesand proteins. A variety of other expression/host systems may beutilized. These systems include but are not limited to microorganismssuch as bacteria transformed with recombinant bacteriophage, plasmid orcosmid DNA expression vectors; yeast transformed with yeast expressionvectors; insect cell systems infected with virus expression vectors(e.g., baculovirus); plant cell systems transfected with virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with bacterial expression vectors (e.g., Tior pBR322 plasmid); or animal cell systems. Mammalian cells useful inrecombinant protein productions include but are not limited to VEROcells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells(such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and293 cells. Mammalian host cells may be preferred when post-translationalmodifications such as glycosylation and polypeptide processing areimportant for activity. Mammalian expression allows for the productionof secreted or soluble polypeptides which may be recovered from thegrowth medium.

Using an appropriate host-vector system, ActRIIB5 proteins andpolypeptides are produced recombinantly by culturing a host celltransformed with an expression vector containing the nucleic acidmolecules of the present invention under conditions allowing forproduction. Transformed cells can be used for long-term, high-yieldprotein production. Once such cells are transformed with vectors thatcontain selectable markers as well as the desired expression cassette,the cells can be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The selectable marker isdesigned to allow growth and recovery of cells that successfully expressthe introduced sequences. Resistant clumps of stably transformed cellscan be proliferated using tissue culture techniques appropriate to thecell line employed. An overview of expression of recombinant proteins isfound in Methods of Enzymology, v. 185, Goeddell, D. V., ed., AcademicPress (1990).

In some cases, such as in expression using procaryotic systems, theexpressed polypeptides of this invention may need to be “refolded” andoxidized into a proper tertiary structure and disulfide linkagesgenerated in order to be biologically active. Refolding can beaccomplished using a number of procedures well known in the art. Suchmethods include, for example, exposing the solubilized polypeptide to apH usually above 7 in the presence of a chaotropic agent. The selectionof chaotrope is similar to the choices used for inclusion bodysolubilization, however a chaotrope is typically used at a lowerconcentration. Exemplary chaotropic agents are guanidine and urea. Inmost cases, the refolding/oxidation solution will also contain areducing agent plus its oxidized form in a specific ratio to generate aparticular redox potential which allows for disulfide shuffling to occurfor the formation of cysteine bridges. Some commonly used redox couplesinclude cysteine/cystamine, glutathione/dithiobisGSH, cupric chloride,dithiothreitol DTT/dithiane DTT, and 2-mercaptoethanol (bME)/dithio-bME.In many instances, a co-solvent may be used to increase the efficiencyof the refolding. Commonly used cosolvents include glycerol,polyethylene glycol of various molecular weights, and arginine.

The proteins and polypeptides of the present can be synthesized insolution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart and Young (supra); Tam et al., J Am Chem Soc, 105:6442, (1983);Merrifield, Science 232:341-347 (1986); Barany and Merrifield, ThePeptides, Gross and Meienhofer, eds, Academic Press, New York, 1-284;Barany et al., Int J Pep Protein Res, 30:705

It is necessary to purify the proteins and polypeptides of the presentinvention. Protein purification techniques are well known to those ofskill in the art. These techniques involve, at one level, the crudefractionation of the proteinaceous and non-proteinaceous fractions.Having separated the peptide polypeptides from other proteins, thepeptide or polypeptide of interest can be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of polypeptides or thepresent invention are ion-exchange chromatography, exclusionchromatography; polyacrylamide gel electrophoresis; isoelectricfocusing. A particularly efficient method of purifying peptides is fastprotein liquid chromatography or even HPLC. The term “isolatedpolypeptide” or “purified polypeptide” as used herein, is intended torefer to a composition, isolatable from other components, wherein thepolypeptide is purified to any degree relative to itsnaturally-obtainable state. A purified polypeptide therefore also refersto a polypeptide that is free from the environment in which it maynaturally occur. Generally, “purified” will refer to a polypeptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a peptide or polypeptidecomposition in which the polypeptide or peptide forms the majorcomponent of the composition, such as constituting about 50%, about 60%,about 70%, about 80%, about 90%, about 95% or more of the proteins inthe composition.

Various methods for quantifying the degree of purification ofpolypeptide will be known to those of skill in the art in light of thepresent disclosure. These include, for example, determining the specificbinding activity of an active fraction, or assessing the amount ofpeptide or polypeptide within a fraction by SDS/PAGE analysis. Apreferred method for assessing the purity of a polypeptide fraction isto calculate the binding activity of the fraction, to compare it to thebinding activity of the initial extract, and to thus calculate thedegree of purification, herein assessed by a “-fold purificationnumber.” The actual units used to represent the amount of bindingactivity will, of course, be dependent upon the particular assaytechnique chosen to follow the purification and whether or not thepolypeptide or peptide exhibits a detectable binding activity.

Various techniques suitable for use in purification will be well knownto those of skill in the art. These include, for example, precipitationwith ammonium sulphate, PEG, antibodies (immunoprecipitation) and thelike or by heat denaturation, followed by centrifugation; chromatographysteps such as affinity chromatography (e.g., Protein-A-Sepharose), ionexchange, gel filtration, reverse phase, hydroxylapatite and affinitychromatography; isoelectric focusing; gel electrophoresis; andcombinations of these techniques. As is generally known in the art, itis believed that the order of conducting the various purification stepsmay be changed, or that certain steps may be omitted, and still resultin a suitable method for the preparation of a substantially purifiedpolypeptide.

Antibodies

The present invention further includes antibodies which specificallybind to the ActRIIB5 receptor polypeptides of the present invention. Asused herein the term “specifically binds” refers to antibodies having abinding affinity (Ka) for ActRIIB5 polypeptides of 10⁶ M⁻¹ or greater.As used herein, the term “antibody” refers to intact antibodiesincluding polyclonal antibodies (see, for example Antibodies: ALaboratory Manual, Harlow and Lane (eds), Cold Spring Harbor Press,(1988)), and monoclonal antibodies (see, for example, U.S. Pat. Nos. RE32,011, 4,902,614, 4,543,439, and 4,411,993, and Monoclonal Antibodies:A New Dimension in Biological Analysis, Plenum Press, Kennett, McKearnand Bechtol (eds.) (1980)). As used herein, the term “antibody” alsorefers to a fragment of an antibody such as F(ab), F(ab′), F(ab′)₂, Fv,Fc, and single chain antibodies which are produced by recombinant DNAtechniques or by enzymatic or chemical cleavage of intact antibodies.The term “antibody” also refers to bispecific or bifunctionalantibodies, which are an artificial hybrid antibody having two differentheavy/light chain pairs and two different binding sites. Bispecificantibodies can be produced by a variety of methods including fusion ofhybridomas or linking of Fab′ fragments. (See Songsivilai et al, Clin.Exp. Immunol. 79:315-321 (1990), Kostelny et al., J. Immunol.148:1547-1553 (1992)). As used herein the term “antibody” also refers tochimeric antibodies, that is, antibodies having a human constantantibody immunoglobin domain coupled to one or more non-human variableantibody immunoglobin domain, or fragments thereof (see, for example,U.S. Pat. No. 5,595,898 and U.S. Pat. No. 5,693,493). Antibodies alsorefers to “humanized” antibodies (see, for example, U.S. Pat. No.4,816,567 and WO 94/10332), minibodies (WO 94/09817), maxibodies, andantibodies produced by transgenic animals, in which a transgenic animalcontaining a proportion of the human antibody producing genes butdeficient in the production of endogenous antibodies are capable ofproducing human antibodies (see, for example, Mendez et al., NatureGenetics 15:146-156 (1997), and U.S. Pat. No. 6,300,129). The term“antibodies” also includes multimeric antibodies, or a higher ordercomplex of proteins such as heterdimeric antibodies, and anti-idiotypicantibodies. “Antibodies” also includes anti-idiotypic antibodies. Theantibodies against ActRIIB5 can be used, for example, to identify andquantitate ActRIIB5 in vitro and in vivo.

Pharmaceutical Compositions

Pharmaceutical compositions containing the ActRIIB5 polypeptides andproteins of the present invention are also provided. Such compositionscomprise a therapeutically or prophylactically effective amount of thepolypeptide in admixture with pharmaceutically acceptable materials, andphysiologically acceptable formulation materials. The pharmaceuticalcomposition may contain formulation materials for modifying, maintainingor preserving, for example, the pH, osmolarity, viscosity, clarity,color, isotonicity, odor, sterility, stability, rate of dissolution orrelease, adsorption or penetration of the composition. Suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates, other organic acids); bulking agents (such asmannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides and other carbohydrates (such as glucose, mannose, ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring; flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides(preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18^(th) Edition, A. R. Gennaro,ed., Mack Publishing Company, 1990).

The optimal pharmaceutical composition will be determined by one skilledin the art depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. See for example,Remington's Pharmaceutical Sciences, supra. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the polypeptide. For example, suitablecompositions may be water for injection, physiological saline solutionfor parenteral administration.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Other exemplary pharmaceutical compositions comprise Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichmay further include sorbitol or a suitable substitute therefore. In oneembodiment of the present invention, compositions may be prepared forstorage by mixing the selected composition having the desired degree ofpurity with optional formulation agents (Remington's PharmaceuticalSciences, supra) in the form of a lyophilized cake or an aqueoussolution. Further, the therapeutic composition may be formulated as alyophilizate using appropriate excipients such as sucrose.

The formulations can be delivered in a variety of methods, for example,by inhalation therapy, orally, or by injection. When parenteraladministration is contemplated, the therapeutic compositions for use inthis invention may be in the form of a pyrogen-free, parenterallyacceptable aqueous solution comprising the desired polypeptide in apharmaceutically acceptable vehicle. A particularly suitable vehicle forparenteral injection is sterile distilled water in which a polypeptideis formulated as a sterile, isotonic solution, properly preserved. Yetanother preparation can involve the formulation of the desired moleculewith an agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (polylactic acid, polyglycolic acid), beads, orliposomes, that provides for the controlled or sustained release of theproduct which may then be delivered via a depot injection. Hyaluronicacid may also be used, and this may have the effect of promotingsustained duration in the circulation. Other suitable means for theintroduction of the desired molecule include implantable drug deliverydevices.

In another aspect, pharmaceutical formulations suitable for injectableadministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions. In anotherembodiment, a pharmaceutical composition may be formulated forinhalation. Inhalation solutions may also be formulated with apropellant for aerosol delivery. In yet another embodiment, solutionsmay be nebulized. Pulmonary administration is further described in PCTApplication No. PCT/US94/001875, which describes pulmonary delivery ofchemically modified proteins.

It is also contemplated that certain formulations may be administeredorally. In one embodiment of the present invention, molecules that areadministered in this fashion can be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. For example, a capsule may be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the therapeutic molecule. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders may also beemployed. Pharmaceutical compositions for oral administration can alsobe formulated using pharmaceutically acceptable carriers well known inthe art in dosages suitable for oral administration. Such carriersenable the pharmaceutical compositions to be formulated as tablets,pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions,and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate. Dragee cores may be used in conjunction with suitablecoatings, such as concentrated sugar solutions, which may also containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for product identification or to characterizethe quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally also includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving polypeptides in sustained-or controlled-delivery formulations. Techniques for formulating avariety of other sustained- or controlled-delivery means, such asliposome carriers, bio-erodible microparticles or porous beads and depotinjections, are also known to those skilled in the art. See for example,PCT/US93/00829 that describes controlled release of porous polymericmicroparticles for the delivery of pharmaceutical compositions.Additional examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices may includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al., Biopolymers, 22:547-556 (1983), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15:167-277, (1981); Langer et al., Chem. Tech., 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomes, which can be prepared by any ofseveral methods known in the art. See e.g., Eppstein et al., PNAS (USA),82:3688 (1985); EP 36,676; EP 88,046; EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically must be sterile. This may be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using this method may be conducted eitherprior to or following lyophilization and reconstitution. The compositionfor parenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

An effective amount of a pharmaceutical composition to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which thepolypeptide is being used, the route of administration, and the size(body weight, body surface or organ size) and condition (the age andgeneral health) of the patient. Accordingly, the clinician may titer thedosage and modify the route of administration to obtain the optimaltherapeutic effect. A typical dosage may range from about 0.1 mg/kg toup to about 100 mg/kg or more, depending on the factors mentioned above.Polypeptide compositions may be preferably injected or administeredintravenously. Long-acting pharmaceutical compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation. Thefrequency of dosing will depend upon the pharmacokinetic parameters ofthe polypeptide in the formulation used. Typically, a composition isadministered until a dosage is reached that achieves the desired effect.The composition may therefore be administered as a single dose, or asmultiple doses (at the same or different concentrations/dosages) overtime, or as a continuous infusion. Further refinement of the appropriatedosage is routinely made. Appropriate dosages may be ascertained throughuse of appropriate dose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g. orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, intralesional routes, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, or intraperitoneal; as wellas intranasal, enteral, topical, sublingual, urethral, vaginal, orrectal means, by sustained release systems or by implantation devices.Where desired, the compositions may be administered by bolus injectionor continuously by infusion, or by implantation device. Alternatively oradditionally, the composition may be administered locally viaimplantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. Wherean implantation device is used, the device may be implanted into anysuitable tissue or organ, and delivery of the desired molecule may bevia diffusion, timed-release bolus, or continuous administration.

In some cases, the ActRIIB5 polypeptides of the present invention can bedelivered by implanting certain cells that have been geneticallyengineered, using methods such as those described herein, to express andsecrete the polypeptide. Such cells may be animal or human cells, andmay be autologous, heterologous, or xenogeneic. Optionally, the cellsmay be immortalized. In order to decrease the chance of an immunologicalresponse, the cells may be encapsulated to avoid infiltration ofsurrounding tissues. The encapsulation materials are typicallybiocompatible, semi-permeable polymeric enclosures or membranes thatallow the release of the protein product(s) but prevent the destructionof the cells by the patient's immune system or by other detrimentalfactors from the surrounding tissues.

ActRIIB5 gene therapy in vivo is also envisioned wherein a nucleic acidmolecule encoding ActRIIB5, or a variant or derivative of ActRIIB5 isintroduced directly into the subject. For example, a nucleic acidsequence encoding an ActRIIB5 is introduced into target cells via localinjection of a nucleic acid construct with or without an appropriatedelivery vector, such as an adeno-associated virus vector. Alternativeviral vectors include, but are not limited to, retroviruses, adenovirus,herpes simplex, virus and papilloma virus vectors. Physical transfer ofthe virus vector may be achieved in vivo by local injection of thedesired nucleic acid construct or other appropriate delivery vectorcontaining the desired nucleic acid sequence, liposome-mediatedtransfer, direct injection (naked DNA), or microparticle bombardment(gene-gun).

Uses of ActRIIB5 Compositions

The present invention provides methods and compositions for reducing orneutralizing the amount or activity of myostatin, activin A, or GDF-11in vivo and in vitro by contacting the proteins with an ActRIIB5protein. The Examples below demonstrate that the ActRIIB5 proteins havea high affinity for myostatin, activin A, and GDF-11, and are capable ofreducing and inhibiting the biological activities of myostatin, activinA and GDF-11. The Examples demonstrate that ActRIIB5 have a higheractivity compared with ActRIIB-ECD as demonstrated by the IC₅₀ values inExample 3, and the biological response in animals is superior for theActRIIB5 animals compared with the ActRIIB-ECD animals as demonstratedin Examples 5 and 6.

In one aspect, the present invention provides methods and reagents fortreating myostatin-related and/or activin A related disorders in asubject in need thereof by administering an effective dosage of anActRIIB5 composition to the subject. As used herein the term “subject”refers to any animal, such as mammals including humans.

The compositions of the present invention have been shown to increaselean muscle mass as a percentage of body weight and decreases fat massas percentage of body weight in animal models as shown in the Examplesbelow.

The disorders that can be treated by an ActRIIB5 composition include butare not limited to various forms of muscle wasting, as well as metabolicdisorders such as diabetes and related disorders, and bone degenerativediseases such as osteoporosis. Muscle wasting disorders includedystrophies such as Duchenne's muscular dystrophy, progressive musculardystrophy, Becker's type muscular dystrophy, Dejerine-Landouzy musculardystrophy, Erb's muscular dystrophy, and infantile neuroaxonal musculardystrophy. Additional muscle wasting disorders arise from chronicdiseases or disorders such as amyotrophic lateral sclerosis, congestiveobstructive pulmonary disease, cancer, AIDS, renal failure, organatrophy, androgen deprivation, and rheumatoid arthritis.

Over-expression of myostatin may contribute to cachexia, a severe muscleand fat wasting syndrome. In one example, serum and intramuscularconcentrations of myostatin-immunoreactive protein was found to beincreased in men exhibiting AIDS-related muscle wasting and wasinversely related to fat-free mass (Gonzalez-Cadavid et al., PNAS USA95: 14938-14943 (1998)). Myostatin levels have also been shown toincrease in response to burns injuries, resulting in a catabolic muscleeffect (Lang et al, FASEB J 15, 1807-1809 (2001)). Additional conditionsresulting in muscle wasting may arise from inactivity due to disabilitysuch as confinement in a wheelchair, prolonged bed rest due to stroke,illness, spinal chord injury, bone fracture or trauma, and muscularatrophy in a microgravity environment (space flight). For example,plasma myostatin immunoreactive protein was found to increase afterprolonged bed rest (Zachwiej a et al. J Gravit Physiol. 6(2):11 (1999).It was also found that the muscles of rats exposed to a microgravityenvironment during a space shuttle flight expressed an increased amountof myostatin compared with the muscles of rats which were not exposed(Lalani et al., J. Endocrin 167 (3):417-28 (2000)).

In addition, age-related increases in fat to muscle ratios, andage-related muscular atrophy appear to be related to myostatin. Forexample, the average serum myostatin-immunoreactive protein increasedwith age in groups of young (19-35 yr old), middle-aged (36-75 yr old),and elderly (76-92 yr old) men and women, while the average muscle massand fat-free mass declined with age in these groups (Yarasheski et al. JNutr Aging 6(5):343-8 (2002)). In addition, myostatin has now been foundto be expressed at low levels in heart muscle and expression isupregulated after cardiomyocytes after infarct (Sharma et al., J CellPhysiol. 180 (1):1-9 (1999)). Therefore, reducing myostatin levels inthe heart muscle may improve recovery of heart muscle after infarct.

Myostatin also appears to influence metabolic disorders including type 2diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, andobesity. For example, lack of myostatin has been shown to improve theobese and diabetic phenotypes of two mouse models (Yen et al. supra). Ithas been shown in the Examples below that administering AAV-ActRIIB5vectors increases the muscle to fat ratio in an animal, in particularfor obese animal models. Therefore, decreasing fat composition byadministering the compositions of the present invention will improvediabetes, obesity, and hyperglycemic conditions in animals. In additionthe Examples below and FIG. 4B demonstrates that compositions containingActRIIB5 may decrease food intake in obese individuals.

In addition, increasing muscle mass by reducing myostatin levels mayimprove bone strength and reduce osteoporosis and other degenerativebone diseases. It has been found, for example, that myostatin-deficientmice showed increased mineral content and density of the mouse humerusand increased mineral content of both trabecular and cortical bone atthe regions where the muscles attach, as well as increased muscle mass(Hamrick et al. Calcif Tissue Int 71(1):63-8 (2002)). In addition, theActRIIB compositions of the present invention can be used to treat theeffects of androgen deprivation such as androgen deprivation therapyused for the treatment of prostate cancer.

The present invention also provides methods and compositions forincreasing muscle mass in food animals by administering an effectivedosage of the ActRIIB5 proteins to the animal. Since the matureC-terminal myostatin polypeptide is identical in all species tested,ActRIIB5 proteins would be expected to be effective for increasingmuscle mass and reducing fat in any agriculturally important speciesincluding cattle, chicken, turkeys, and pigs.

The ActRIIB5 proteins and compositions of the present invention alsoantagonizes the activity of activin A. Activin A is known to beexpressed in certain types of cancers, particularly gonadal tumors suchas ovarian carcinomas, and to cause severe cachexia. (Ciprano et al.Endocrinol 141 (7):2319-27 (2000), Shou et al., Endocrinol 138(11):5000-5 (1997); Coerver et al, Mol Endocrinol 10(5):534-43 (1996);Ito et al. British J Cancer 82(8):1415-20 (2000), Lambert-Messerlian, etal, Gynecologic Oncology 74 91):93-7 (1999). Example 4 below shows thatexpression of activin A in the animal models results in a severecachexia. Expression of the ActRIIB5/Fc in the animals counters thatcachexia, as shown in Examples 5 and 6. Overexpression of myostatin isalso thought to contribute to cachexia, as described above. Thereforethe compositions can be used to treat conditions related to activin Aoverexpression, as well as myostatin overexpression, such as cachexiafrom certain cancers and the treatment of certain gonadal type tumors.

The compositions of the present invention may be used alone or incombination with other therapeutic agents to enhance their therapeuticeffects or decrease potential side effects. These properties includeincreased activity, increased solubility, reduced degradation, increasedhalf-life, reduced toxicity, and reduced immunogenicity. Thus thecompositions of the present invention are useful for extended treatmentregimes. In addition, the properties of hydrophilicity andhydrophobicity of the compounds of the invention are well balanced,thereby enhancing their utility for both in vitro and especially in vivouses. Specifically, compounds of the invention have an appropriatedegree of solubility in aqueous media that permits absorption andbioavailability in the body, while also having a degree of solubility inlipids that permits the compounds to traverse the cell membrane to aputative site of action, such as a particular muscle mass.

In addition, the ActRIIB5 proteins and polypeptides of the presentinvention are useful for detecting and quantitating myostatin, activinA, or GDF-11 in any number of assays. In general, the ActRIIB5polypeptides of the present invention are useful as capture agents tobind and immobilize myostatin, activin A, or GDF-11 in a variety ofassays, similar to those described, for example, in Asai, ed., Methodsin Cell Biology, 37, Antibodies in Cell Biology, Academic Press, Inc.,New York (1993). The polypeptides may be labeled in some manner or mayreact with a third molecule such as an antibody which is labeled toenable myostatin to be detected and quantitated. For example, apolypeptide or a third molecule can be modified with a detectablemoiety, such as biotin, which can then be bound by a fourth molecule,such as enzyme-labeled streptavidin, or other proteins. (Akerstrom, JImmunol 135:2589 (1985); Chaubert, Mod Pathol 10:585 (1997)).

The invention having been described, the following examples are offeredby way of illustration, and not limitation.

Example I Isolation of cDNA and Expression in Cells

The cDNA of the novel human activin type IIB receptor was isolated froma cDNA library of human testis origin (Clontech, Inc.) according to thefollowing protocol. Primers for the N-terminal and the C-terminal of thehuman activin IIB receptor (SEQ ID NO: 4) were generated and PCR wasperformed using these primers against templates from human cDNAlibraries. PCT was performed using the GC-RICH PCR System (Roche, cat#2140306). Both N and C terminal PCR products were digested withPvuII/EcoRI and subcloned into pcDNA3.1-H isA vector (Invitrogen,Carlsbad, Calif.) to make a full length clone. After sequencing a numberof PCR products, a cDNA clone from the human testes cDNA library wasidentified as a novel N-terminal splice variant receptor. Thepolynucleotide sequence of this receptor, designated human activin typeIIB5 receptor (ActRIIB5). The cDNA clone of this receptor was missing152 nucleotide bases that correspond to the entire Exon-4 in thewild-type human activin type IIB receptor gene. The truncation of exon-4in the splice variant resulted in the deletion of the amino acidsequence that spans the transmembrane region as well as in a frame shiftleading to an early translational termination. The amino acid sequenceof the splice variant receptor contains most of the extracellulardomain, encoded by exons 1, 2 and 3 of the wild-type human activin typeIIB receptor, and an additional tail region of 36 amino acids resultingfrom the frame shift. The amino acid sequence is set forth in SEQ ID NO:2. The C terminal sequence is set forth in SEQ ID NO: 3. Due to the lackof transmembrane region, the ActRIIB5 encodes a soluble form of activintype IIB receptor. Transfection of the ActRBII5 cDNA in cells led to theexpression of secreted, instead of membrane-bound, form of the receptorprotein.

Example 2 Expression of ActRIIB5

cDNA encoding ActRIIB5 was cloned into a mammalian pDC323 or pDC324vectors (Bianchi et al, Biotech and Bioengineering, Vol. 84(4):439-444(2003)) and expressed in a 293T cell line. To generate the Fc fusions,polynucleotides encoding the ActRIIB5 (SEQ ID NO:1) were cloned adjacentto polynucleotides encoding the (Gly)₈ linker sequence adjacent topolynucleotides encoding the human IgG1 Fc into a pDSRa vector(described in WO/9014363, herein incorporated by reference).Polynucleotides encoding ActRIIB-ECD (amino acids 1-124 of SEQ ID NO: 5)were cloned adjacent to polynucleotides encoding the human IgG1 Fc intoa pDSRa vector (no linker). These constructs were transfected into astable CHO cell line. The soluble receptor-Fc fusions expressed wereused for the side-by-side in vitro testing described below.

For the in vivo animal experiments described in Example 4 below, the PCRproducts generated as described above were digested with NheI/SalI andsubcloned into an AAV-Fc vector at the same sites. The AAV-Fc vectorallows for transfer of the ActRIIB5 gene into an animal for expressionin vivo.

Example 3 In Vitro Activities

HuActRIIB5/Fc and HuActRIIB-ECD/Fc were generated as described above.The ability the ActRIIB5 receptor to inhibit the binding of each of thethree ligands myostatin, activin A, and GDF-11 to the activin IIBreceptor was tested using a cell based activity assay as describedbelow.

C2C12 Cell Based Activity Assay

A myostatin/activin/GDF-11-responsive reporter cell line was generatedby transfection of C2C12 myoblast cells (ATCC No: CRL-1772) with apMARE-luc construct. The pMARE-luc construct was made by cloning twelverepeats of the CAGA sequence, representing the myostatin/activinresponse elements (Dennler et al. EMBO 17: 3091-3100 (1998)) into apLuc-MCS reporter vector (Stratagene cat #219087) upstream of the TATAbox. The myoblast C2C12 cells naturally express myostatin/activin/GDF-11receptor activin receptor IIB on its cell surface. Whenmyostatin/activinA/GDF-11 binds the cell receptors, the Smad pathway isactivated, and phosphorylated Smad binds to the response element(Macias-Silva et al. Cell 87:1215 (1996)), resulting in the expressionof the luciferase gene. Luciferase activity is then measured using acommercial luciferase reporter assay kit (cat #E4550, Promega, Madison,Wis.) according to manufacturer's protocol. A stable line of C2C12 cellsthat had been transfected with pMARE-luc (C2C12/pMARE clone #44) wasused to measure activity according to the following procedure. Reportercells were plated into 96 well cultures. Screening using dilutions ofeach type of soluble receptor was performed with the concentration fixedat 4 nM myostatin, 20 nM activin, and 4 nM GDF-11. Myostatin, activinand GDF-11 were each pre-incubated with the soluble receptors at severalconcentrations. Myostatin/activin/GDF-11 activity was measured bydetermining the luciferase activity in the treated cultures. The IC₅₀values were for the determined for each soluble receptor as set out inTable 1 below.

TABLE 1 activin A neutralizing activity Soluble receptor protein IC50(nM) vs. 20 nM activin huActRIIB5/Fc 156.2 huActRIIB-ECD/Fc 339.6myostatin-neutralizing activity soluble receptor protein IC50 (nM) vs. 4nM myostatin huActRIIB5/Fc 29.72 huActRIIB-ECD/Fc 51.06GDF-11-neutralizing activity soluble receptor protein IC50 (nM) vs. 4 nMGDF-11 huACtRIIB5/Fc 90.6 huActRIIB-ECD/Fc 89.88

The table above shows that the soluble receptors can block myostatinsignaling through its receptor but also activin A and GDF-11 signaling.

BIAcore® Assay

Blocking assays were carried out using immobilized human ActRIIB-ECD/Fc(R&D Systems, Minneapolis, Mn.) on a CM5 chip (Biacore, Inc.,Piscataway, N.J.) in the presence and absence of each of the two solublereceptors ActRIIB-ECD/Fc and ActRIIB5/Fc using the BIAcore® assay systemaccording to the manufacturer's instructions.

100% myostatin binding signal was determined in the absence of receptorin solution. Various concentrations of the soluble receptors werediluted in sample buffer and incubated with 4 nM myostatin before beinginjected over the receptor surface. Since only free myostatin moleculeswere able to bind to the chip, a decreased binding response withincreasing concentration of the receptors indicated binding of thereceptors to myostatin in solution. Plotting the binding signal vs.concentration of soluble receptor, ActRIIB-ECD/Fc and ActRIIB5/Fc werecalculated to have an EC₅₀ of approximately 18 nM and 7 nM respectively.The comparison between the two receptors is shown in FIG. 1.

Example 4 Activin a Over-Expression in C57B1/6 Mice

To explore the postnatal role of activin in postnatal animals, activin Awas overexpressed in mice using AAV mediated gene transfer. Aged-matchedyoung adult (5-week-old) female C57B1/6 mice (Charles Riverlaboratories, Wilmington, Mass.) were separated into two weight-balancedgroups (n=6/group), which were subsequently injected via portal veinwith either AAV-activin A or AAV-empty vector (control) at 1×10¹³pfu/mouse. The effects on body weight and body composition wereanalyzed. AAV-activin A transduced group showed a drastic reduction inbody weight compared to the control mice transduced with AAV-emptyvector. Within 2 weeks post AAV injection, the activin A-transducedgroup became so severely cachectic that their average body weight wasonly about ½ of that of empty vector-transduced control group. Necropsyrevealed that AAV-activin A administration resulted a dramatic depletionby approximately 60% of lean body mass, skeletal muscle mass and fatmass. In addition, the activin-transduced mice also showed severewasting of organs as indicated by significantly reduced organ weightssuch as liver and heart.

An additional experiment using a reduced amount (1×10¹² pfu/mouse) ofAAV-Activin A virus was performed. The results showed a reduction inbody weight and lean body mass resulting from activin-transduction butthe effects were less dramatic as compared to the initial experimentusing AAV-activin A at a higher dose (1×10¹³ pfu/mouse). Thisdemonstrates that the postnatal cachectic effect of activin A isdose-dependent.

Example 5 Anabolic Effect of AAV-ActRIIB5 in C57B1/6 Mice

Age-matched (5-week-old) C57B1/6 male mice were divided into 5 groups(n=10 per group). AAV viral particles were packaged and titered prior toinjection as follows: AAV-empty, AAV-activin A, AAV-ActRIIB5/Fc,AAV-ActRIIB-ECD/Fc, and AAV-ProMyo/Fc, wherein AAV-ProMyo stands forpropeptide of myostatin. Each of the above AAV viruses were injected at8×10¹² pfu/mouse except for AAV-activin A, of which an reduced amount ofviral particles at 1×10¹² pfu/mouse was injected (n=10/group). The viralparticles were injected via the portal vein. Body weights weredetermined every other day. The results are shown in FIG. 2.AAV-ActRIIB5/Fc group and the AAV-ActRIIB-ECD/Fc group developedincreased body weights compared to the AAV-Vector control group, as wellas increased body weight compared to the AAV-ProMyo/Fc group. Comparingthe two soluble receptor groups, the AAV-ActRIIB5/Fc group showed thegreatest amount of increase in body weight gain. In contrast, theAAV-Vector control group showed a dramatic decrease in body weights incomparison to the AAV-Vector control group.

At seven weeks post viral injection, body weight changes of individualgroups were plotted as percentage of that of the control group(AAV-empty vector group). The AAV-ActRIIB5/Fc group showed the highestaverage body weight increase over control, approximately 25%, comparedwith 21% body weight increase for the ActRIIB-ECD/Fc group. TheAAV-ActRIIB5/Fc group and the AAV-ActRIIB-ECD/Fc group showed bodyweight increases greater than that elicited by the ProMyo/Fc group ofapproximately 16%. In contrast, AAV-activin group had a significant dropin body weight by 19%. A comparison of these changes is shown in FIG. 3.

One-month post viral injection, lean body mass in each group of ten micewas determined using nuclear magnetic resonance (NMR) by measuring bodycomposition of live mice. At the same time, the body fat content of themice in each group was determined. The measurements were taken on livemice using the EchoMRI 2003 (Echo Medical Systems, Houston, Tx). EchoMRI2004 is a whole body composition analyzer that measures the masses offat and lean tissues in live animals using NMR technology. The averagepercentage of lean mass and fat as percentage of body weight for eachgroup of 10 mice is presented in Table 2 below.

TABLE 2 Fat (% body weight) lean mass (% body weight)AAV-promyostatin/Fc 9.11 90.33 AAV-Activin A 10.21 87.92 AAV-empty 11.7686.74 AAV-ActRIIB5/Fc 7.82 91.05 AAV-ActRIIB-ECD/ 8.51 90.18 Fc

As can be seen from Table 2 above, the AAV-ActRIIB5/Fc group of miceshowed the smallest percentage of body fat, and the largest percentageof lean mass for all of the groups after one month. This data shows thatActRIIB5/Fc is effective in enhancing body weight, lean body mass anddecreasing fat mass in the animals tested.

In a related experiment, the five groups of ten mice per group weretested for gripping strength using a Columbia Instruments meter, model1027 dsm (Columbus, Ohio). The results were averaged for each group. TheAAV-promyostatin/Fc group averaged a gripping strength compared with theAAV-empty control mice was about 21% for the promyostatin/Fc group,about 31% for the ActRIIB-ECD/Fc group and about 33% for the ActRIIB5/Fcgroup of mice. The increase in gripping strength measured was about 46%for the promyostatin/Fc group, about 56% for the ActRIIB-ECD/Fc group,and about 60% for the ActRIIB5/Fc group.

Example VI Changes in Body Weight and Composition in Ay Obese Mice

Two groups of Ay Obese mice (Jackson Laboratories, Bar Harbor, Me.) of11 animals each (8 animals per group at the termination of theexperiment) were injected with an AAV-empty vector and anAAV-ActRIIB5/Fc vector respectively. The viruses were injected at 8×10¹²pfu/mouse into the portal vein of each mouse. The mice were thenmonitored for changes in body weight, food intake, lean muscle mass andfat mass over a three month period post injection. Food intake wasdetermined by weighing the remaining uneaten food in the mouse cage on adaily basis and calculating the weekly intake. The lean muscle mass andfat mass were determined by NMR as described above. The results of theexperiments are shown in FIGS. 4 and 5. FIG. 4A shows a decrease in bodyweight and FIG. 4B shows a decrease in weekly food intake in theAAV-ActRIIB5/Fc mice compared with the control mice. FIG. 5A showsincrease in lean mass, as determined by NMR for the AAV-ActRIIB5/Fc,while FIG. 5B shows a large decrease of fat mass for the AAV-ActRIIB/Fccompared to the control mice, by approximately 50%.

At the termination of the experiment, the mice were sacrificed andexamined for internal changes. The livers of the AAV-ActRIIB5/Fc treatedmice were compared with those treated with AAV-empty control. Visualinspection of the livers of the AAV-empty treated mice and theAAV-ActRIIB5/Fc treated mice showed that the livers of the controlAAV-empty mice contained fat deposits within the livers, whereas theAAV-ActRIIB5/Fc treated mice were free of fat deposits. Therefore, theexpression of the ActRIIB5/Fc in the Ay mice corrected the fatty liverswhich characterize the Ay obese mice, as well as caused a decrease inoverall body weight, a decrease in amount of food consumed, an increasein lean muscle mass and large decrease in fat mass.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide selected from the group consisting of: (a) apolynucleotide having the polynucleotide sequence set forth in SEQ IDNO: 1 or its complement; (b) a polynucleotide encoding a polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO: 2; and (c)the polynucleotide sequence that hybridizes to either (a) or (b) underconditions of moderate stringency in about 50% formamide, 6×SSC at about42° C. and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS, andwherein polypeptide encoded comprises a C terminal having an amino acidsequence set forth in SEQ ID NO: 3, and wherein the polypeptide iscapable of binding myostatin, activin A, or GDF-11. (d) thepolynucleotide sequence that hybridizes to either (a) or (b) underconditions of moderate stringency in about 50% formamide, 6×SSC at about42° C. and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS, andwherein polypeptide encoded comprises a C terminal having an amino acidsequence at least 80% identical to SEQ ID NO: 3, and wherein thepolypeptide is capable of binding myostatin, activin A, or GDF-11.
 2. Anisolated nucleic acid molecule comprising a polynucleotide encoding apolypeptide having an amino acid sequence at least 80% identical to theamino acid sequence set forth in SEQ ID NO: 2, wherein thepolynucleotide comprises a C terminal having the amino acid sequence setforth in SEQ ID NO:
 3. 3. The isolated nucleic acid sequence of claim 2,wherein the polynucleotide comprises a C terminal having an amino acidsequence at least 80% identical to SEQ ID NO:
 3. 4. The isolated nucleicacid molecule of claim 1, wherein the nucleic acid molecule furthercomprises polynucleotides encoding least one heterologous protein inframe with the polynucleotides encoding an activin type IIB5 receptor.5. The isolated nucleic acid molecule of claim 4, wherein heterologousprotein is an Fc polypeptide.
 6. The isolated nucleic acid molecule ofclaim 5, wherein the Fc is attached by a linker peptide.
 7. An isolatednucleic acid molecule comprising a polynucleotide consisting of thesequence set forth in SEQ ID NO:
 1. 8. The nucleic acid molecule of anyone of claims 1 through 7, wherein the polynucleotide is operably linkedto a transcriptional or translational regulatory sequence.
 9. Thenucleic acid molecule of claim 8 wherein the transcriptional ortranslational sequence comprises a transcriptional promoter or enhancer.10. A recombinant vector that directs the expression of the nucleic acidmolecule of claim
 1. 11. An isolated protein comprising an activin typeIIB5 receptor polypeptide, wherein the polypeptide is selected from thegroup consisting of: (a) a polypeptide consisting of the amino acidsequence set forth in SEQ ID NO: 2; (b) a polypeptide consisting of anamino acid sequence having at least 80% identity to SEQ ID NO: 2,wherein the polypeptide is capable of binding myostatin, activin A orGDF-11; (c) the polypeptide of (b), wherein the C terminal of thepolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3;and wherein the polypeptide is capable of binding myostatin, activin A,or GDF-11; and (d) the polypeptide of (b), wherein the C terminal of thepolypeptide comprises an amino acid sequence having at least 80%identity to SEQ ID NO: 3, and wherein the polypeptide is capable ofbinding myostatin, activin A, or GDF-11.
 12. An isolated proteincomprising an activin type IIB5 receptor polypeptide, wherein thepolypeptide consists of the amino acid sequence set forth in SEQ ID NO:2.
 13. The protein of claim 12, wherein amino acid residue 64 in SEQ IDNO: 2 is alanine.
 14. An isolated protein comprising a polypeptideencoded by the polynucleotide set forth in SEQ ID NO:
 1. 15. The proteinof claim 11, wherein the polypeptide is fused to at least oneheterologous polypeptide.
 16. The protein of claim 15, wherein theheterologous protein is an Fc polypeptide.
 17. The protein of claim 16,wherein the Fc polypeptide is attached via a linker sequence.
 18. A hostcell genetically engineered to express the nucleic acid molecule ofclaim
 1. 19. The host cell of claim 18 wherein the host cell is amammalian cell.
 20. A host cell genetically engineered to produce theprotein of claim
 11. 21. The host cell of claim 20, wherein the hostcell is a mammalian cell.
 22. A method of producing an activin IIB5receptor polypeptide comprising culturing the host cell of claim 21under conditions promoting expression of the polypeptide, and recoveringthe polypeptide.
 23. A pharmaceutical composition comprising the activintype IIB5 receptor protein of claim 11 in admixture with apharmaceutically acceptable carrier.
 24. A method of inhibitingmyostatin activity in a subject in need thereof comprising administeringa therapeutically effective amount of the composition of claim 23 to thesubject.
 25. A method of increasing lean muscle mass in a subject inneed thereof comprising administering a therapeutically effective amountof the composition of claim 23 to the subject.
 26. A method ofincreasing the ratio of lean muscle mass to fat in a subject in needthereof comprising administering a therapeutically effective amount ofthe composition of claim 23 to the subject.
 27. A method of treating amuscle-wasting disease in a subject suffering from such as diseasecomprising administering a therapeutically effective amount of thecomposition of claim 23 to the subject.
 28. The method of claim 27,wherein the disease is cancer cachexia.
 29. The method of claim 27,wherein the disease selected from muscular dystrophy, amyotrophiclateral sclerosis, congestive obstructive pulmonary disease, chronicheart failure, cancer cachexia, AIDS, renal failure, uremia, rheumatoidarthritis, age-related sarcopenia, organ atrophy, carpal tunnelsyndrome, androgen deprivation, and muscle-wasting due to prolonged bedrest, spinal chord injury, stroke, bone fracture, and aging.
 30. Amethod of treating a metabolic disorder in a subject in need thereofcomprising administering a therapeutically effective amount of thecomposition of claim 23 to the subject.
 31. The method of claim 30,wherein the metabolic disorder is selected from diabetes, obesity,hyperglycemia, and bone loss.
 32. A method of treating a disease inwhich activin is over-expressed in a subject comprising administering atherapeutically effective amount of the composition of claim 23 to thesubject.
 33. The method of claim 30, wherein the disease is cancer. 34.The method of claim 25, wherein the subject is a food animal.
 35. Amethod of treatment of a muscle wasting disorder comprisingadministering the vector of claim 10 to a subject, wherein the vector iscapable of directing expression of ActRIIB5 polypeptides in the subject.36. The method of claim 35 wherein the vector is an AAV vector.